Secretion of factors that mediate host cell invasion and manipulation are key to the success of apicomplexans. However, we still do not know where or how some of these factors are secreted, for example, those from dense granules. Toxoplasma ‘apical annuli’ are small ring structures in the inner membrane complex (IMC) at the lower boundary of the apical cap cisterna. The function and significance of these enigmatic structures have been unknown. Furthermore, the previously identified annuli proteins are associated with the IMC, so it was uncertain if this structure has any interaction with or relevance to the plasma membrane and extracellular environment. We used hyperLOPIT spatial proteomics and proximity labelling to search for plasma membrane-associated proteins at the apical annuli and found four with this conspicuous location. One is a polytopic membrane protein containing LMBR1 domains but of unknown function, and three are Q-SNAREs implicated in vesicle docking. Depletion of each of these annuli proteins results in strong lytic cycle growth phenotypes. Specifically, these mutations manifest as defects in intracellular replication and the delivery of secreted proteins, including GPI-anchored proteins. Our data show that the apical annuli are essential structures to Toxoplasma, that their composition and activity span the plasma membrane, and that they represent additional exocytic sites to the apical complex in tachyzoites. We speculate that the complexity and persistence of the IMC in Toxoplasma has driven the need for apical annuli as further specialised secretion sites in the cell periphery.
